TA systems are common in bacteria. There are eight families of TA systems all of which operate under a similar principle, with a stable toxin and a labile antitoxin prone to proteases that are induced upon stress. Among the TA pair, the MazEF and RelBE systems are the best studied. Genomic analysis revealed MazEF and RelBE paralogs in many Gram+ bacteria. Preliminary studies indicated that the MazF toxin in S. aureus is a specific endoribonuclease that cuts at a VUUV'site where V or V'can be A, C or G. We speculate that it may be feasible to disrupt the binding of the toxin to the antitoxin MazE with small synthetic compounds to unleash the toxicity of MazF, thus representing a new approach for the development of novel antimicrobial compounds. We also examined the specificity of MazF on Mrna cleavage and found that some housekeeping and virulence regulator mRNAs are spared while others such as hla, spa and sigB are easily cleaved. We suspect RNA-binding protein may protect some of these mRNAs. As we already have a few "putative compounds" from our pilot screen, we propose to screen for small synthetic compounds against anti-MazE, using a novel fluorescent DNA-RNA hybrid substrate in the assay. To satisfy the above goal, we have developed the following aims: I) Identification of RNA binding protein(s) that protects selective mRNA (e.g. selective housekeeping gene and sarA) from MazFsa cleavage;II) developing an assay to screen for small molecule(s) that interferes with function or binding of MazEsa to MazFsa. Collectively, these data will highlight the uniqueness of the TA systems in Gram+ bacteria. As therapy options against multidrug resistant S. aureus are limited, our screening assays will provide the initial compounds against MazE for the development of novel antimicrobial therapy against S. aureus